A pyrimidine-requiring mutant isolated from Chinese hamster cell V79 has been tested in various substrates involved in the pyrimidine biosynthetic pathway. According to the growth behavior of the mutant cells, they do not grow in carbamyl phosphate, carbamyl aspartic acid, dihydroorotate, orotic acid, and ortate monophosphate (OMP), but do grow in UMP. The blockage in the pyrimidine pathway seems to involve the enzyme OMP decarboxylase. Whether the genetic defect is a consequence of a mutation in the regulatory gene or in the structural gene is still to be determined by the construction of cell hybrid between pyr and human lymphocytes.